A chemical method for labeling lysine methyltransferase substrates.

Olivier Binda,Krishnan K Palaniappan,Michael Boyce,Or Gozani,Jason S Rush,Carolyn R Bertozzi

doi:10.1002/cbic.201000433

Abstract

Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

Highlights

The latest human genome annotation predicts 52 protein lysine methyltransferases (PKMTs) and 33 protein lysine demethylases (PKDMTs) based on sequence similarities to known catalytic domains
Using varying amounts of 1, ranging from 0 to 100 mm, we found that the optimal concentration of 1 for H3 modification by SETDB1 is 50 mm (Figure 1 B)
We note that our experiment probably overestimates the concentration of analogue needed for optimal activity, as it is likely that only one diastereomer is accepted by SETDB1, as with natural SAM

Summary

A Chemical Method for Labeling Lysine Methyltransferase Substrates

Olivier Binda,[a, b] Michael Boyce,[c] Jason S. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts

Introduction

Results and Discussion

Conclusions